PKU is a human genetic disorder in which the body does not contain or contain in less amounts or less stable forms of the PAH enzyme, necessary to metabolize L-phenylalanine (L-Phe) to L-tyrosine (L-Tyr), but converts phenylalanine instead to phenylpyruvic acid by transamination, however, decarboxylation of L-Phe may also occur. That is, the body of the individual affected with said disease is not able to utilize the essential amino acid phenylalanine. Amino acids are the building of food since the individual does normally not produce the essential amino acids. In “classic phenylketonuria (PKU)” the enzyme that brakes down phenylalanine, PAH, is completely or nearly completely deficient, thus, no conversion to tyrosine takes place. Without this enzyme, phenylalanine and its breakdown chemicals from other enzymeroutes, accumulate in the blood and body tissues (Scriver C R, Kaufman S. Hyperphenylalaninemia: phenylalanine hydroxylase deficiency. In: Scriver C R, Beaudet A L, Valle D, Sly W S, eds. The Metabolic and Molecular bases of Inherited Disease. 8th ed. New York: McGraw-Hill, 2001: 1667-1724).
Further, the disease termed “hyperphenylalaninemia” (HPA) is used to describe the phenomenon of having elevated blood phenylalanine and, normally, include the specific form of PKU. About 98% of patients displaying HPA carry mutations in the phenylalanine hydroxylase (pah) gene, while the remaining 2% mainly affect enzymes involved in the synthesis or regeneration of PAH cofactor, tetrahydrobiopterin (BH4) Scriver C R, Kaufman S. supra). In the following, HPA is used to include all type of diseases or disorders or conditions characterized in having an elevated phenylalanine level. PKU represents a special form of a disorder falling under the general term of HPA, where HPA is due to mutations in the pah gene.
As mentioned before, PKU and HPA are both caused by mutations in the pah gen, More than 60% of mutations associated to PKU are missense mutations (leading to a single amino acid substitution on the protein), and their main effects on PAH function seem to be associated to protein), and their main effects on PAH function seem to be associated to protein misfolding and increased turnover in vivo. Due to defects in converting the amino acid L-Phe to L-Tyr, L-Phe accumulates in the blood and is mainly excreted unchanged in the urine; some is transaminated to phenylpyruvic acid, which may be further metabolized to phenylacetic, phenyllactic and o-hydroxyphenylacetic acids; all are excreted in the urine (Scriver C R, Kaufman S. supra), Waters P J, Hum Mutat 2003; 21(4):357-69; Scriver C R. The PAH gene, phenylketonuria, and a paradigm shift. Hum Mutat 2007.
PKU and most other causes of HPA are inherited in a recessive fashion (Scriver C R, Kaufman S. supra) This means, an affected person inherited two traits for the disorder. A person carrying only one mutation in the phenylalanine hydroxylase gene is called carrier for PKU, carriers do not have symptoms of the disorder. Classic PKU and the other causes of HPA affect about 1 of every 10,000 to 20,000 Caucasian or Oriental births. The incidence in African Americans is far less. These disorders are equally frequent in males and females.
The phenylalanine hydroxylase (PAH, phenylalanine 4-monooxigenase; EC1.14.16.1) is a non-heme iron dependent enzyme that catalyzes the hydroxylation of L-Phe to L-Tyr in the presence of tetrahydrobiopterin (BH4) and molecular dioxygen as cosubstrates, Fitzpatrick P F, Adv. Enzymol. Relat. Areas Mol. Biol. 2000; 74:235-294. In humans, PAH activity is mainly present in liver, and essential to provide L-Tyr for protein and neurotransmitters biosynthesis in liver, and essential to provide L-Tyr for protein and neurotransmitters biosynthesis, besides its involvement in energy metabolism. About 500 disease causing mutations have been described for the PAH gene, see e.g. the phenylalanine hydroxylase locus knowledgebase (PAHdb), at http:www.pandb.mcgill.cs. The molecular mechanisms responsible for the loss-of-function of PAH have pointed towards decreased conformational stability and, in some cases, kinetic abnormalities in the PAH enzyme in vitro and probably in vivo (Waters P J., Hum Mutat 2003; 21(4):357-69; Scriver C R., Hum Mutat 2007; Erlandsen H., et al., Proc Natl Acad Sci USA 2004; 101(48):16903-8).
Restriction on L-Phe intake by using artificial dietary formulations and early diagnosis through newborn screening tests have led to a remarkable success in preventing the major manifestations of the disease, including mental retardation. However, diet therapy has to be maintained “for life”, it is relatively expensive and socially burdening and if not continued it may affect fetus development during pregnancy.
Typically, HPA is associated with mental retardation. Further, untreated children with classical PKU are normal at birth, but fail to attain earlier developmental milestones, develop microcephaly, and demonstrate progressive impairment of cerebral function. Hyperactivity, seizures, and severe mental retardation are major clinical problems later in life. Electroencephalographic abnormalities; “mousy” odor of skin, hair and urine (due to Phenylacetate accumulation); and a tendency to hypopigmentation and eczema complete the devastating clinical picture. With maternal PKU it is essential for women with PKU for the health of their children to maintain low phenylalanine levels before and during pregnancy. Otherwise, as a result, the children may develop congenital heart disease, growth retardation, microcephaly and mental retardation, (Gambol P J., J Pediatr Nurs 2007; 22(2):129-38
Currently, several different strategies to partially or totally substitute low-Phe diet and to treat PKU are investigation. BH4 supplementation has been demonstrated to short and long term reduce-L-Phe levels and increase L-Phe tolerance in mils and severe PKU phenotypes, increasing the L-Phe oxidation in vivo, Blau N, Erlandsen H. Mol Genet Metad 2004; 82(2):101-11, probably due to a multifactor mechanisms involving stabilization of PAH mutant proteins against degradation/inactivation, Erlandsen H, et al., Proc Natl Acad Sci USA 2004; 101(48):16903-8. Large neutral amino acids (LNAA) supplementation is based on the reversal of the L-Phe induced inhibition of the LNAA transport across the blood-brain barrier by the L-type amino acid carrier, Matalon R, et al., Pediatrics 2003; 112(6Pt 2)1570-1574. The above approaches allow to facilitate the restrictive L-Phe diets. Additional approaches like gene-therapy and enzyme replacement therapy are aimed to completely eliminate L-Phe dietary restricition and are currently the subject of intense research (Gamez A, et al., Mol ther 2005; 11(6):986-9 Wang L, et al., Mol Genet Metab 2005; 86(1-2):134-40; Harding C O, et Al., Ther 2006; 13(5):457-62; Ding Z, et al., Gene ther 2006; 13(7):587-93.
The object of the present invention is to provide a new approach to treat PKU and HPA and to provide new compounds useful therefore.